Receptors which are binding sites for proteins and small molecules are attractive targets for pharmacological intervention in disease-related processes. One group that fits this category of receptors is comprised of members of the LRP family. The term LRP is an abbreviation for LDL-Receptor-related Proteins, where the LDL receptors are a group of proteins involved in the binding and transportation of Low-density Lipoprotein (LDL) into cells by endocytosis. Various proteins are considered to be members of the LRP family because of their resemblance to LDL-receptors as well as their resemblance to each other. FIG. 1 shows various members of the LRP family, where different motifs that are held in common are shown for various members. The most important common elements are the YWTD β-propellors, EGF-like domains and LDL receptor-like ligand binding domains. These elements may appear as singular elements or they may comprise multimeric repeats. The members of this family are also characterized by a transmembrane domain that anchors the LRP extracellular portion to a membrane surface as well as an intracellular domain that may interact with cellular proteins. Although the LRP family members are structurally related, the functions they serve in vivo are of a diverse nature that include the uptake of lipoproteins, endocytosis, transcytosis, signal transduction, vitamin and hormonal homeostasis, as well as phagocytosis of necrotic cells (reviewed in Herz and Strickland 2001 J. Clin. Invest. 108:779-784). In conjunction with the various roles that these proteins may be involved in, members of the LRP family recognize a large number of ligands. For instance, one member alone, LRP1, recognizes at least 30 different ligands that in themselves represent several families of proteins. These ligands include lipoproteins, proteinases, proteinase inhibitor complexes, ECM proteins, bacterial toxins, viruses, and various intracellular proteins.
Some of the proteins that bind to members of the LRP family are involved in Wnt signaling. For example, Wnt has been shown to directly interact with one or more of the YWTD domains of the amino (extracellular) portion of LRP5 and LRP6 to induce Wnt signaling. Another example is Dkk, which is believed to bind to different domains of LRP5 and LRP6 (the third and fourth YWTD domains) but nonetheless influences the ability of the first or second domain of LRP5 and LRP6 to bind to Wnt. Other proteins such as Frat 1 (Hay et al. 2005 J Biol Chem 14; 13,616-13,623), Christin/R-spondin proteins (Nam et al., JBC 2006) and connective-tissue growth factor (CTGF) (Mercurio et al., 2003 Development 131; 2137-2147) also interact with the extracellular Domains of LRP5 while Casein kinase I (Davidson et al. 2005 Nature 438; 867-872, Swiatek et al., 2006 J Biol Chem 281; 12,233-12,241), Glycogen synthase kinase 3 (GSK3) (Mi et al., JBC 281; 4787-4794, Zeng et al., Nature 438; 873-877) and Axin (Mao et al., 2001 Mol Cell 7; 801-809) have been shown to interact with the intracellular portion. The ability to bind to a protein may or may not be involved in signal functions of an LRP molecule. For example, the majority of ligands that bind to the multiligand receptor LRP1 are either proteases or molecules associated with the control of proteolytic activity. However, although the LRP1 receptor is not commonly associated with Wnt pathway events, investigations have revealed that under appropriate conditions, truncated versions of LRP1 were able to interact with Frizzled, a major component of the Wnt signaling pathway (Zilberberg et al., 2004 J. Biol. Chem. 279; 17,535-17,542). This interaction is dissimilar to the well characterized system involving interactions of LRP5 and LRP6 and Wnt elements since the effect of both the truncated as well as the full length version of LRP1 is the opposite of the classical LRP5 and LRP6 interactions. The binding of LRP1 to Frizzled represses Wnt signaling instead of inducing it.
Some of the proteins that bind to members of the LRP family are not involved in Wnt signaling. Even with LRP members like LRP5 and LRP6, which are known to play a major part in Wnt signaling, certain ligands that bind to LRP5 and LRP6 have been shown not to affect the Wnt pathway. For instance, Wei et al. have demonstrated that LRP6 mediates the internalization and lethality of anthrax toxin (Cell 124, 1141-1154, Mar. 24, 2006), and the role of LRP5 in cholesterol metabolism is believed to be Wnt independent (Magoori et al., 2003 J. Biol. Chem. 278; 11,331-11,336). With regard to the latter, Fujino et al. (2003 Proc. Nat. Acad. Sci. (USA) 100; 229-234) investigated the metabolic consequences of a genetic ablation of LRP5 and concluded that LRP5 is essential for both normal cholesterol metabolism and glucose-induced insulin secretion. The presence of an LRP5 deficiency in either homozygous (LRP5−/−) or even heterozygous (LRP5+/−) mice resulted in a significant increase in plasma cholesterol levels when the animals were fed a high-fat diet. Although fasted blood glucose and insulin levels were normal in the mutant strains, they showed a defect in glucose tolerance when challenged. These animals also showed impaired clearance of chylomicron remnants and also impaired glucose-induced insulin secretion from the pancreatic islets. The effect of a lack of LRP5 was also tested in a double mutation situation where the mice lacked not only LRP5, but also apoE (Magoori et al. 2003). Although neither condition alone led to changes in cholesterol levels with a normal diet, the double condition led to 60% higher plasma cholesterol levels. At 6 months of age, the double-null mice had also developed severe atherosclerotic lesions that were three times larger than those in knockout mice missing only apoE. The connection between LRP molecules and metabolism is also evidenced by the discovery that certain polymorphisms in the LRP5 gene have been correlated with obesity phenotypes in a family based study (Guo et al., 2006 J. Med. Genet. 43; 798-803). Lastly, a mutation in LRP6 has been correlated to an autosomal dominant defect that results in the expression of phenotypic features associated with metabolic syndrome: hyperlipidemia, hypertension and diabetes (Mani et al., 2007 Science 315; 1278-1282.
There is a distinction between transducer (LRP5 and LRP6 receptors) and non-transducer multi-ligand receptors (non-LRP5 and non-LRP6 receptors). In the case of a non-transducing receptor, the term “multi-ligand” encompasses broad specificity, as in the case of a receptor that takes up different monosaccharides. In this case, essentially the same effect (transport) is carried out by the receptor for a variety of different ligands where each internalized ligand is then recognized and processed according to its specific chemical nature. On the other hand, for multi-ligand signal receptors, another layer of complexity is observed where different domains participate in different reactions. In the case of signal transducers, the ligand per se is not the target of further downstream actions. In fact, as a rule, it is not even internalized. Thus, the specificity of the signal transduction is entirely the result of the specificity of the transducer. This means that if two different ligands elicit two different downstream responses, there must be a difference, however subtle, in the way they trigger the transducer after binding.
With regard to the LRP5 and LRP6 receptor, it is quite obvious that the extracellular and intracellular domains must by necessity have different ligands and different functions. Even within the extracellular portion itself, there will be differentiation of function for the different domains of LRP5 and LRP6. For example, the first two YWTD domains in the extracellular portion of LRP5 and LRP6 are involved in binding Wnt and transmitting a signal, while the third and fourth domains are sites for binding of a completely different protein, Dkk, and a subsequent dampening of Wnt signaling. Remarkably, LRPs combine features of both types of multi-ligand receptors since they can function both as an internalizer and as a transducer.
Although domains of functional and structural similarity can be identified through amino acid alignments, the ability of such analogues to carry out different functions is a product of their fine differences. As described in the review article by Herz and Stickland that was cited earlier: “Crystallographic and nuclear magnetic resonance studies of individual repeats have revealed that the sequence variability in short loop regions of each repeat results in a unique surface contour surface and charge density for each repeat.” In summary, even when a collection of repeated sequences are able to form similar structures, the particular nature of the amino acids on their exposed surfaces will still dictate the ability to bind different ligands. Interactions between individual amino acids will also cause differences in the overall structure where cavities in comparable domains may be slightly larger or smaller due to small scale attractive or repulsive forces. This can be seen in the studies of LRP5 and LRP6 where the size of the opening in the β-propeller of a YWTD repeat region is different from one domain to another. More importantly, as described in section 4.2 of U.S. Patent Application No. 20050196349, identification of amino acid residues that are important for Dkk binding was carried out by alanine scanning. A comparison of nucleic acid and amino acid sequences shows that there are substitutions of different amino acids at analogous sites (U.S. patent application Ser. No. 11/598,916) within these cavities thereby differentiating the degree of affinity between molecules that may be similar in size but different in terms of polarity and/or charge with regard to binding to each of the domains.
In the previously cited patent applications, the use of a detailed three-dimensional model of the LRP5 receptor allowed a virtual screening of a library of compounds for predicting molecules that would fit into a binding domain of LRP5. As disclosed in U.S. Patent Application No. 20050196349, a variety of different biological results can be seen when these compounds are tested with in vitro assays. Looking at Table II, it can be seen that some of the compounds (Group 1) are toxic as exemplified by compounds IIC5, IIIC6, and IIIC12 which reduced basal expression to 26%, 0% and 10%, respectively. Not surprisingly, further experiments showed a lack of stimulation when Wnt was added. Other compounds such as IIC6, IIC18 and IIC19 were not intrinsically toxic, since they maintained or even stimulated basal level expression. However, in this group of compounds (Group 2), the addition of Wnt showed no stimulation, indicating an inability to respond to Wnt in the presence of these compounds. A third class of compounds (Group 3) showed a normal level of response to the addition of Wnt compared to the no drug control, but showed a diminished effect of inhibition by Dkk. For instance, IIC8 (NCI 39914) allowed essentially the same level of stimulation by Wnt as in its absence (1227 compared to 1000 in the absence of drug), but when Dkk was added, the amount of activity was only reduced to 476. The control showed a shift of 1000 to 100 by the addition of Dkk. Even more strikingly, IIIC3 (NCI 8642) shows almost the same amount of activity in the presence of Dkk as in its absence, demonstrating that the binding of this molecule can lead to a block in Wnt suppression by Dkk. There is even one compound, IIC9, that represents a fourth group of compounds that was able to reduce the amount of Wnt stimulation, but instead of showing Dkk suppression, Wnt activity was stimulated three fold by the presence of Dkk. Thus, it can be seen that binding to LRP5 and LRP6 does not necessarily lead to a single phenotype in these assays.
There are a variety of reasons why these different effects may be seen. For instance, although one particular domain was chosen for the selection of a ligand from the library, a biological assay may reveal that the affinity of the compound is higher for a different (but similar) domain on the target protein. There is also the possibility of mimicry, where the binding of the compound to the Dkk site on LRP5 and LRP6 in itself emulates the same effect seen by binding of the true ligand and leads to “Dkk-like” suppression of Wnt activity in the absence of Dkk. It is also natural to assume, especially in the case of a multi-ligand receptor, that allosteric effects are possible that influence separate binding events at sites away from where the drug itself may bind.
In the previously disclosed applications, molecules with properties described for the third class of compounds (Group 3) were tested for various biological activities besides the LEF reporter system in order to test for a biological effectiveness for disease processes. Among the assays described in these disclosures were those related to bone formation and remodeling as witnessed by assays for osteoblast differentiation in U.S. Patent Application No. 20050196349. Two compounds from this group, IIC8 and IIIC3, were tested for an additional property, the ability to block the binding of sclerostin, a protein which has previously been shown to have an effect similar to that of Dkk in being able to block Wnt signaling. Experimental results showed a direct correlation where increased amounts of these compounds resulted in decreased binding of sclerostin-AP. These compounds as well as other similar compounds were also tested for effects on bone growth via calavarial bone formation, β-catenin activity and viability in various tumor cell lines, tumor induction in a mouse model, as well as metabolic effects such as cholesterol and glucose metabolism (U.S. patent application Ser. No. 11/598,916). The potential use of pharmaceutical compositions for altering the activity of LRP5 in a subject has been described in U.S. Patent Application No. 20030181660 (hereby incorporated by reference) with specific application to diseases such as diabetes, autoimmune diseases, viral infections, osteoporosis and metabolic disorders, as well as diseases that involve or affect endocytosis, antigen presentation, cytokine clearance or inflammation. However, their approach was directed towards a different level, where they taught the use of compounds to regulate the level of expression of LRP5. In contrast, the methods described in U.S. Patent Application No. 20050196349 have been directed towards the identification of compounds that interact with the LRP5 and LRP6 protein or associated proteins.
A similar program of virtual screening followed by binding studies was carried out for compounds predicted to bind to Disheveled, another member of the Wnt signaling pathway (U.S. patent application Ser. No. 11/097,518). In this case, molecules of interest were followed with testing for effects on embryogenesis.